ProjectSummary Organoidsaremini-??tissuestructuresthatarerevolutionizinginvitrostudiesduetothefactthattheyare derivedfromnormalordiseasedtissuesfromhumandonors,inducedpluripotentstemcells(iPSCs),andnearly every model organism. The use of organoids in disease modeling has become a powerful method to replicate pathophysiology usingstraightforward cell culture conditions. Virtually every tissue type now has an in vitro organoid correlate. As thename implies, organoids are representations of tissue layers (typically epithelium) thathaveaspecificfunctioninanorganism.Theyaretypicallyspherical,andarestemcelldriven,giventhem potentialtoproduceallthedifferentiatedcelltypesfoundinanygiventissue.Thishasledtoinaccurateresults, failure in predicting drug efficacy, and loss of millions of dollars and man hours following lines of research basedonartifact.Challengesassociatedwithrapidlyestablishingorganoidculturesarelargelyassociatedwith initiating a clonal colony from asingle cell, tracking colonygrowth and inducing differentiation processes. In addition, systems supporting organoid culture must allow time-??course based analysis of physiological phenotypes. Cell Microsystems has developed the CellRaft Technology, a microwell array-??based platform where single cells can be seeded in small culture chambers, grown into clonal colonies and tracked over time usingvirtuallyanyimagingmodality.Thesecapabilitieshaveshowninitialpromiseinaddressingmanyofthe challengesassociatedwithorganoidculture.ScottMagness,PhDoftheUniversityofNorthCarolinaatChapel Hill has published methods using CellRaft Arrays for establishing, growing and analyzing organoids derived from various enteric stem cells. The research proposed here builds on both innovative array designs and materialsbyCellMicrosystems,aswellastheworkflowsestablishedbyDr.Magness.Underthiscollaborative project,wewilldevelopanovelCellRaftArray,the3D-??CytoSortArray,withlargermicrowellsthanarecurrently manufactured(500micronsand1millimetersquare)withgreaterdepth(300micronsinsteadofthestandard80 microns)tofacilitatethegrowth,differentiationandanalysisofvarioustypesoforganoid.Thesearrayswillbe validated for use with Cell Microsystems AIR? System, an automated platform for the imaging, sorting and isolation of cells or colonies from the CellRaft Array. This instrument will allow not only the imaging of organoids for temporal analysis, but also isolation of organoids for downstream molecular analysis via next generation sequencing or other molecular analysis modality. Dr. Magness? team will translate their current organoid culture methods to the new, larger 3D-??CellRaft Array. Successful recapitulation of their previously published data using the new array and the AIR? System will serve as an initial validation of the product?s performance as a platform for broad organoid culture methods. Pending completion of the Specific Aims proposedhere,aPhaseIIprojectwillbeproposedthatwillexpandtherangeoforganoidtypeswiththegoalof expandingtheutilityoftheAIRSystemsoftwarefororganoid-??specificanalysis.